Customized Consensus Spectral Library Building for Untargeted Quantitative Metabolomics Analysis with Data Independent Acquisition Mass Spectrometry and MetaboDIA Workflow.

Data independent acquisition-mass spectrometry (DIA-MS) coupled with liquid chromatography is a promising approach for rapid, automatic sampling of MS/MS data in untargeted metabolomics. However, wide isolation windows in DIA-MS generate MS/MS spectra containing a mixed population of fragment ions together with their precursor ions. This precursor-fragment ion map in a comprehensive MS/MS spectral library is crucial for relative quantification of fragment ions uniquely representative of each precursor ion. However, existing reference libraries are not sufficient for this purpose since the fragmentation patterns of small molecules can vary in different instrument setups. Here we developed a bioinformatics workflow called MetaboDIA to build customized MS/MS spectral libraries using a user's own data dependent acquisition (DDA) data and to perform MS/MS-based quantification with DIA data, thus complementing conventional MS1-based quantification. MetaboDIA also allows users to build a spectral library directly from DIA data in studies of a large sample size. Using a marine algae data set, we show that quantification of fragment ions extracted with a customized MS/MS library can provide as reliable quantitative data as the direct quantification of precursor ions based on MS1 data. To test its applicability in complex samples, we applied MetaboDIA to a clinical serum metabolomics data set, where we built a DDA-based spectral library containing consensus spectra for 1829 compounds. We performed fragment ion quantification using DIA data using this library, yielding sensitive differential expression analysis.

Comparison of swept source optical coherence tomography and spectral domain optical coherence tomography in polypoidal choroidal vasculopathy.

BACKGROUND: Swept source optical coherence tomography (SS-OCT, Topcon Medical System, Japan) is known to have longer wavelength than spectral domain OCT (SD-OCT, Spectralis, Heidelberg Engineering, Germany), allowing a deeper penetration into retina and choroidal layers. This objective of this study was to compare the visibility of retinal and choroidal features in polypoidal choroidal vasculopathy (PCV) using SS-OCT and SD-OCT. DESIGN: This study employs prospective comparative observational case series in Singapore National Eye Center. PARTICIPANTS: There were 20 eyes (20 patients) with PCV confirmed with indocyanine green angiogram. METHODS: Six pre-specified OCT parameters (presence of polyps, sharp pigment epithelial detachment [PED] peak, notched PED and visibility of full maximum height of PED, inner segment/outer segment [IS/OS] line and choroid-scleral interface [CSI]) were graded using SS-OCT and SD-OCT. MAIN OUTCOME MEASURES: The Kappa statistics between the two imaging modalities were calculated. RESULTS: Both SS-OCT and SD-OCT were able to detect polypoidal lesions in the majority of eyes (90% and 85%, respectively). However, SS-OCT had better detection for CSI and IS/OS lines (CSI: 80% vs 45%, P = 0.05; IS/OS line: 65% vs 45%, P = 0.34). For sharp PED peak, notched PED, ability to visualize the full PED height and retinal pigment epithelial line, both OCT machines were able to detect in ≥80% of the eyes. CONCLUSION: In conclusion, SS-OCT and SD-OCT appeared to be similarly effective at detecting most features associated with PCV. However, SS-OCT is more superior in detecting the CSI.

Posterior segment findings of ocular cytomegalovirus infection in immunocompetent patients.

PURPOSE: We sought to investigate and describe the clinical spectrum of posterior segment abnormalities in immunocompetent patients presenting with CMV-associated anterior uveitis. METHODS: This was a prospective study conducted at the Singapore National Eye Centre, a tertiary referral centre, from August 2010 to June 2011. Eleven eyes of eleven patients with CMV anterior uveitis confirmed by polymerase chain reaction on aqueous humor sampling were recruited based on the study criteria. Patients were recruited from a single uveitis specialist clinic and underwent aqueous humor sampling and fluorescein and indocyanine green angiography as well as optical coherence tomography. They were further evaluated by the Infectious Disease physician for immunocompetence. RESULTS: Mean presenting visual acuity was logMAR 0.35 ± 0.29. The main presenting complaints were blurring of vision, eye redness, and pain. Anterior chamber cellular activity was present in all cases. Fine diffuse keratic precipitates (KPs) were present in 10 eyes, and the remaining one eye had mutton fat KPs. Iris changes were present in three eyes. Intraocular pressure (IOP) was elevated in nine eyes (mean presenting IOP was 40.2 ± 16.8 mmHg). In the posterior segment, none of the eyes had evidence of retinitis or hemorrhage. Posterior segment abnormalities were present in six eyes (macular edema, disc leakage, epiretinal membrane, phlebitis). Eight eyes also had prolonged arm to retina time (mean 24.8 ± 10.6 s) on fluorescein angiography. Indocyanine green angiography was unremarkable. CONCLUSION: Posterior segment manifestations can be seen in a proportion of immunocompetent patients with CMV anterior uveitis. The underlying mechanism remains to be determined.

Multimodal Imaging-Based Phenotyping of a Singaporean Hospital-Based Cohort of High Myopia Patients.

Purpose: To assess the effect of axial length (AL) on the prevalence of pathologic myopia (PM) and associated myopic features in a Singaporean hospital-based cohort of patient with high myopia (HM). Methods: In total, 923 HM eyes from 495 individuals were recruited from the Myopic and Pathologic Eyes in Singapore (MyoPES) cohort and underwent ocular biometry, fundus photography, fundus autofluorescence, and swept-source optical coherence tomography (SS-OCT). Images were analyzed for the presence of myopic macular degeneration (MMD), myopic choroidal neovascularization (mCNV), myopic traction maculopathy (MTM), peripapillary atrophy (PPA), myopic tilted disc, posterior staphyloma (PS), dome-shaped macula (DSM), vitremacular adhesions (VMA), and the epiretinal membrane (ERM). Eyes were stratified into quartiles based on ALs to determine cut-off values to perform comparisons between shorter-length and longer-length groups. A χ2-test was done to determine the difference in the prevalence of pathologies between groups. Results: Overall, mean AL was 29.2 ± 2.2 mm (range 25.0-36.7 mm). Myopic macular degeneration, PPA, myopic tilted disc, and ERM have AL threshold of ≥27.5 mm, whereas MTM has an AL threshold of ≥29.0 mm. We found that there was a significantly higher prevalence of MMD (88.2 vs. 49.4%; p < 0.001), PPA (98.1 vs. 80.1%; p < 0.001), myopic tilted disc (72.7 vs. 50.2%; p < 0.001), and ERM (81.4 vs. 17.3%; p = 0.003) in eyes with AL ≥ 27.5 mm vs. eyes without AL <27.5 mm. Prevalence of MTM (34.7 vs. 32.1%; p < 0.001), mCNV (17.4 vs. 12.1%; p = 0.03), PS (43.4 vs. 34.7%; p = 0.012), DSM (21.3 vs. 13.2%; p = 0.002), and VMA (5.9 vs. 2.6%; p = 0.014) in eyes with AL ≥ 29.0 mm compared with AL < 29.0 mm. Conclusion: Our study describes the overall prevalence of PM and related pathologies among patients with HM in our hospital-based cohort. Longer eyes even among HM eyes had a significantly higher prevalence of PM-associated pathologies studied. This supports the premise that eyes with longer AL, even among HM eyes may be at greater risk of vision-threatening changes and therefore merit regular follow-up.

New Concepts in Polypoidal Choroidal Vasculopathy Imaging: A Focus on Optical Coherence Tomography and Optical Coherence Tomography Angiography.

Polypoidal choroidal vasculopathy (PCV) is a variant of neovascular age-related macular degeneration. It is characterized by polypoidal dilatations at the terminus of branching vascular network located beneath the retinal pigment epithelium. These polypoidal lesions are best visualized on indocyanine green angiography. With recent advances in ocular imaging, optical coherence tomography (OCT) and OCT angiography (OCTA) have been increasingly used to aid in the diagnosis and monitoring of treatment responses in PCV. This review provides a summary of the current status of various imaging modalities in PCV, with special focus on OCT and OCTA.

Advances in Retinal Imaging and Applications in Diabetic Retinopathy Screening: A Review.

Rising prevalence of diabetes worldwide has necessitated the implementation of population-based diabetic retinopathy (DR) screening programs that can perform retinal imaging and interpretation for extremely large patient cohorts in a rapid and sensitive manner while minimizing inappropriate referrals to retina specialists. While most current screening programs employ mydriatic or nonmydriatic color fundus photography and trained image graders to identify referable DR, new imaging modalities offer significant improvements in diagnostic accuracy, throughput, and affordability. Smartphone-based fundus photography, macular optical coherence tomography, ultrawide-field imaging, and artificial intelligence-based image reading address limitations of current approaches and will likely become necessary as DR becomes more prevalent. Here we review current trends in imaging for DR screening and emerging technologies that show potential for improving upon current screening approaches.

Identification of imaging features that determine quality and repeatability of retinal capillary plexus density measurements in OCT angiography.

PURPOSE: Optical coherence tomography angiography (OCT-A) potentially allows for rapid and non-invasive quantification of retinal capillary plexus density in various disease states. This study aims to identify the key features that influenced the repeatability of OCT-A measurements. METHODS: We obtained OCT-A images on two separate visits in 44 healthy eyes from 44 subjects, each imaged with using the Topcon DRI OCT Triton imaging system. The parafoveal vessel density within a 1.5 mm radius centred over the fovea was obtained with the built-in tool for the superficial and deep retinal plexuses. Repeatability of vessel density was determined by intraclass correlation (ICC) and mean variation. We evaluated several image parameters to determine their influence on the repeatability of vessel density measurement in each of the two capillary plexuses. RESULTS: The mean age of the subjects was 70.2±9.2 years, with 64% males. Mean parafoveal vessel density measurements for the first and second visits were 53.3±11.1 and 53.3±10.3 for the superficial plexus and 27.3±8.59 and 27.0±8.78 for the deep plexus. ICC analyses demonstrated that high fine vessel visibility, the absence of motion artefact and software-derived image quality score of 60 or above were necessary to obtain a good (ICC>0.6) or excellent (ICC>0.75) repeatability. CONCLUSIONS: Our study identified the imaging parameters that determined the repeatability of quantitative retinal vessel density measurements. These findings have implications in determining if OCT-A images can be used to accurately evaluate serial changes in retinal vessel density.

Diabetic macular oedema.

Diabetic macular oedema, characterised by exudative fluid accumulation in the macula, is the most common form of sight-threatening retinopathy in people with diabetes. It affects one in 15 people with diabetes resulting in more than 20 million cases worldwide. Few epidemiological studies have been done to specifically investigate risk factors for diabetic macular oedema, although poor glycaemic and blood pressure control are associated with the presence and development of the disorder. The pathophysiological processes begin with chronic hyperglycaemia, and interplay between vascular endothelial growth factor (VEGF) and inflammatory mediators. Non-invasive imaging using optical coherence tomography has allowed clinicians to detect mild levels of diabetic macular oedema in order to monitor progress and guide treatment. Although focal or grid laser photocoagulation was the traditional mode of treatment, intraocular pharmacotherapy with anti-VEGF agents is now the standard of care. However, these therapies are expensive and resource intensive. Emerging therapeutic strategies include improving efficacy and duration of VEGF suppression, targeting alternative pathways such as inflammation, the kallikrein-kinin system, the angiopoietin-Tie2 system, and neurodegeneration, and using subthreshold and targeted laser therapy. Ongoing research should lead to improvements in screening, diagnosis, and management of diabetic macular oedema.

Peripapillary choroidal thickness assessed using automated choroidal segmentation software in an Asian population.

AIMS: To objectively quantify the thickness of peripapillary choroid using spectral-domain optical coherence tomography (SD-OCT) with enhanced depth imaging (EDI) followed by a novel automated choroidal segmentation software in Asian eyes and to evaluate its systemic and ocular determinants. METHODS: We recruited 520 subjects (1040 eyes) from the Singapore Malay Eye Study, a cross-sectional population-based study. Subjects underwent standardised detailed ophthalmic examination including SD-OCT (Spectralis) with EDI for measurement of peripapillary choroidal thickness (PPCT). RESULTS: The mean age of the subjects was 66.7±10.4 years (range 47-88 years) and the mean spherical equivalent was -0.01±2.28 D (range -18.50 to +7.00 D). The intra-session repeatability of PPCT measurements at four quadrants using automated choroidal segmentation software was excellent (intraclass correlation coefficient 0.9998-0.9999). The overall mean PPCT was 136.2±56.8 µm. Peripapillary choroid showed geographical differences among the four quadrants, being thickest in the superior quadrant (150.5±59.6 µm), followed by the nasal (143.5±58.4 µm) and temporal quadrants (139.4±68.9 µm), and thinnest in the inferior quadrant (111.3±51.7 µm). Among the range of ocular and systemic factors studied, shorter axial length (p=0.002), younger age (p=0.018), lower triglyceride level (p=0.015) and the presence of diabetes (p=0.036) were the only significant predictors of thicker peripapillary choroid. CONCLUSIONS: Using novel automated choroidal segmentation software, we provide reliable objective measurements of PPCT in a population-based setting. Shorter axial length, younger age, lower triglyceride levels and the presence of diabetes are the factors independently associated with thicker PPCT. These factors should be taken into consideration when interpreting Spectralis EDI SD-OCT-based PPCT measurements in clinics.

Prevalence and determinants of undiagnosed diabetic retinopathy and vision-threatening retinopathy in a multiethnic Asian cohort: the Singapore Epidemiology of Eye Diseases (SEED) study.

OBJECTIVE: To determine the prevalence and risk factors of undiagnosed diabetic retinopathy (DR), in particular vision-threatening DR (VTDR) in a multiethnic Asian cohort. DESIGN: A population-based survey of 3353 Chinese, 3280 Malays and 3400 Indians (73.6% response) aged 40-80 years residing in Singapore. Diabetes mellitus (DM) was defined as random glucose ≥11.1 mmol/L, use of diabetic medication or a previous physician diagnosis. DR severity was graded from retinal photographs following the modified Airlie House classification. VTDR was defined as the presence of severe non-proliferative DR (NPDR), proliferative DR (PDR) or clinically significant macular oedema (CSMO), using the Eye Diseases Prevalence Research Group definition. Participants were deemed 'undiagnosed' if they reported no prior physician diagnosis in structured interviews, in those with the condition. RESULTS: Of 10 033 participants, 2376 had DM (23.7%), of which 805 (33.9%) had DR. Among 2376 with DM, 11.1% (n=263) were undiagnosed. Among 805 with DR, 671 (83.3%) were undiagnosed. Among 212 with VTDR, 59 (27.3%) were undiagnosed. In multivariate models, factors associated with undiagnosed VTDR were higher low-density lipoprotein (LDL) cholesterol (OR=1.53, 95% CI 0.99 to 2.35, p=0.05) and absence of visual impairment or blindness in any eye in terms of best-corrected vision OR=3.00, 95% CI 1.47 to 6.11, p=0.003). CONCLUSIONS: In this community, a quarter with VTDR is undiagnosed, and 8 in 10 with any DR are undiagnosed, compared with only 1 in 10 with DM undiagnosed. These findings suggest that screening for diabetes is successful, while screening for DR is currently inadequate in our population. Public health strategies to aid early diagnosis of DR in Singapore are urgently warranted to reduce blindness due to diabetes.

Is aspirin intake associated with early age-related macular degeneration? The Singapore Indian Eye Study.

BACKGROUND/AIMS: To examine the relationship between aspirin intake and early age-related macular degeneration (AMD) among an Asian population. METHODS: A population based cross sectional study of 3207 ethnic Indians aged 40 years or older residing in Singapore. AMD signs were graded from retinal images following the modified Wisconsin grading system. Information on aspirin intake was obtained from a standardised questionnaire. RESULTS: The prevalence of early AMD was 5.6%. Aspirin use was reported by 11.4% of participants. Early AMD signs were present among 10.9% of aspirin users and 4.9% of non-aspirin users (p<0.001). After adjusting for potential confounders, including age, smoking and previous cataract surgery, aspirin use was associated with early AMD (OR 1.50; 95% CI: 1.01 to 2.22). The association weakened and was not significant after additional adjustment for cardiovascular disease (OR 1.38; 95% CI 0.89 to 2.14). In stratified analysis, aspirin use was significantly associated with early AMD in participants with (adjusted OR 2.64, 95% CI 1.31 to 5.36) but not without (OR 0.73; 95% CI 0.36 to 1.51) a history of cardiovascular disease (interaction term, p=0.011). CONCLUSIONS: Aspirin intake overall was not associated with early AMD in this sample of Asian Indians, but in those with a history of cardiovascular disease the association between aspirin intake and early AMD might warrant further investigation.

Myopia-related fundus changes in Singapore adults with high myopia.

PURPOSE: To examine the pattern of myopia-related macular and optic disc changes in Singapore adults with high myopia (spherical equivalent ≤-6.00 diopters). DESIGN: Asian adults with high myopia from 3 population-based surveys. METHODS: Adults 40 years and older (n = 359) with high myopia were pooled from 3 population-based surveys in Singapore Asians: (1) the Singapore Prospective Study Program (SP2, n = 184); (2) the Singapore Malay Eye Study (SiMES, n = 98); and (3) the Singapore Indian Eye Study (SINDI, n = 77). All study participants underwent standardized refraction and fundus photography, and SiMES and SINDI subjects also completed ocular biometry measurements. Myopia-related macular (posterior staphyloma, lacquer cracks, Fuchs spot, myopic chorioretinal atrophy, and myopic choroidal neovascularization) and optic disc (optic nerve head tilt, optic disc dimensions, and peripapillary atrophy) changes were evaluated. RESULTS: The most common myopia-related macular finding in adults with high myopia was staphyloma (23%), followed by chorioretinal atrophy (19.3%). There were few cases of lacquer crack (n = 6, 1.8%), T-sign (n = 6, 1.8%), retinal hemorrhage (n = 3, 0.9%), active myopic choroidal neovascularization (n = 3, 0.9%), and no case of Fuchs spot. The most common disc finding associated with high myopia was peripapillary atrophy (81.2%), followed by disc tilt (57.4%). Staphyloma and chorioretinal atrophy increased in prevalence with increasing age, increasing myopic refractive error, and increasing axial length (all P < .001). Ethnicity comparisons demonstrated the highest proportion of staphyloma (P = .04) among Malays, the highest proportion of peripapillary atrophy (P = .01) and disc tilt (P < .001) among Chinese, and the largest cup-to-disc ratio (P < .001) among Indians. CONCLUSIONS: Staphyloma and chorioretinal atrophy lesions were the most common fundus findings among Asian adults with high myopia. In this population, tilted discs and peripapillary atrophy were also common, while choroidal neovascularization and Fuchs spot were rare. In contrast with Singapore teenagers, in whom tilted disc and peripapillary atrophy were common while staphyloma and chorioretinal atrophy were rare, pathologic myopia appears to be dependent on the duration of disease and, thus, age of the individual.

Automatic detection of the macula in retinal fundus images using seeded mode tracking approach.

The macula is the part of the eye responsible for central high acuity vision. Detection of the macula is an important task in retinal image processing as a landmark for subsequent disease assessment, such as for age-related macula degeneration. In this paper, we have presented an approach to automatically determine the macula centre in retinal fundus images. First contextual information on the image is combined with a statistical model to obtain an approximate macula region of interest localization. Subsequently, we propose the use of a seeded mode tracking technique to locate the macula centre. The proposed approach is tested on a large dataset composed of 482 normal images and 162 glaucoma images from the ORIGA database and an additional 96 AMD images. The results show a ROI detection of 97.5%, and 90.5% correct detection of the macula within 1/3DD from a manual reference, which outperforms other current methods. The results are promising for the use of the proposed approach to locate the macula for the detection of macula diseases from retinal images.

Hyperopic refractive error and shorter axial length are associated with age-related macular degeneration: the Singapore Malay Eye Study.

PURPOSE: To describe the association between refractive errors, ocular biometry, and age-related macular degeneration (AMD) in an Asian Malay population in Singapore. METHODS: A population-based study of 3280 Malay individuals aged 40 to 80 years was conducted in Singapore. Early- and late-AMD signs were graded from retinal photographs according to the Wisconsin grading system. Autorefraction, followed by subjective refraction, was performed to obtain spherical equivalent refraction (SER) in diopters, with emmetropia defined as SER -0.5 to +0.5 D, hyperopia as > +0.5 D, and myopia as < -0.5 D. Partial coherence laser interferometry was used to measure axial length, anterior chamber depth, and corneal curvature. The association between refractive status, ocular biometry and the prevalence of both early and late AMD were analyzed. RESULTS: Hyperopic refractive error (odds ratio [OR] 1.54; 95% confidence interval [CI] 1.00-2.36; compared with myopia, P = 0.05), shorter axial length (OR, 1.91; CI, 1.05-3.46, comparing 1st vs. 4th quartiles; P = 0.03), and steeper corneal curvature (OR, 1.93; CI, 1.16-3.20, comparing 1st vs. 4th quartiles, P = 0.01) were significantly associated with early AMD, after adjustment for age, sex, smoking, education, height, and systolic blood pressure. Each diopter increase in hyperopic refraction and each millimeter decrease in axial length was associated with an 8% (OR, 1.08; CI, 1.01-1.16; P = 0.03) and 29% (OR, 1.29; CI, 1.06-1.57; P = 0.01) increased risk of early AMD, respectively. No significant association was noted of refractive error and ocular biometry with late AMD. CONCLUSIONS: Hyperopic refractive error and shorter axial length are associated with early AMD in Asian eyes.